Compositions and methods for identifying and targeting cancer cells of alimentary canal origin

ABSTRACT

Screening and diagnostic reagents, kits and methods for metastatic colorectal cancer or primary and/or metastatic stomach or esophageal cancer are disclosed. Vaccines compositions and methods of for treating and preventing metastatic colorectal cancer or primary and/or metastatic stomach or esophageal cancer are disclosed.

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/819,254 filed Mar. 27, 2001, which claims the benefit of U.S.Provisional Application No. 60/192,229 filed Mar. 27, 2000, each ofwhich is incorporated herein by reference in its entirety.

This application is also related to U.S. Pat. No. 5,518,888, issued May21, 1996, U.S. Pat. No. 5,601,990 issued Feb. 11, 1997, U.S. Pat. No.6,060,037 issued Apr. 26, 2000, U.S. Pat. No. 5,962,220 issued Oct. 5,1999, and U.S. Pat. No. 5,879,656 issued Mar. 9, 1999, which are eachincorporated herein by reference and U.S. patent application Ser. No.09/180,237 filed Mar. 12, 1997, which is incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to in vitro diagnostic methods fordetecting cancer cells of the alimentary canal, particularly primary andmetastatic stomach and esophageal cancer and metastatic colorectalcancer, and to kits and reagents for performing such methods. Thepresent invention relates to compounds and methods for in vivo imagingand treatment of tumors originating from the alimentary canal,particularly primary and metastatic stomach and esophageal tumors andmetastatic colorectal tumors. The present invention relates to methodsand compositions for making and using targeted gene therapy, antisenseand drug compositions. The present invention relates to prophylactic andtherapeutic vaccines against cancer cells of the alimentary canal,particularly primary and metastatic stomach and esophageal cancer andmetastatic colorectal cancer and compositions and methods of making andusing the same.

BACKGROUND OF THE INVENTION

There is a need for reagents, kits and methods for screening, diagnosingand monitoring individuals with cancer originating from the alimentarycanal, particularly primary and metastatic stomach and esophageal cancerand metastatic colorectal cancer. There is a need for reagents, kits andmethods for identifying and confirming that a cancer of unknown originis originating from the alimentary canal and for analyzing tissue andcancer samples to identify and confirm cancer originating from thealimentary canal and to determine the level of migration of such cancercells. There is a need for compositions which can specifically targetcolorectal, stomach and esophageal cancer cells. There is a need forimaging agents which can specifically bind to colorectal, stomach andesophageal cancer cells. There is a need for improved methods of imagingcolorectal, stomach and esophageal cancer cells. There is a need fortherapeutic agents which can specifically bind to colorectal, stomachand esophageal cancer cells. There is a need for improved methods oftreating individuals who are suspected of suffering from primary and/ormetastatic stomach or esophageal cancer or metastatic colorectal cancer.There is a need for vaccine composition to treat colorectal, stomach andesophageal cancer. There is a need for vaccine composition to treat andprevent metastasized bolorectal, stomach and esophageal cancer. There isa need for therapeutic agents which can specifically deliver genetherapeutics, antisense compounds and other drugs to colorectal, stomachand esophageal cancer cells.

SUMMARY OF THE INVENTION

The invention further relates to in vitro methods of determining whetheror not an individual has cancer originating from the alimentary canal,particularly primary and metastatic stomach and esophageal cancer andmetastatic colorectal cancer. The present invention relates to in vitromethods of examining samples of non-colorectal tissue and body fluidsfrom an individual to determine whether or not CDX1, which is expressedby normal colon cells and by colorectal, stomach and esophageal tumorcells, is being expressed by cells in samples other than colon. Thepresence of CDX1 protein or of the CDX1 gene transcript in samplesoutside the colorectal track is indicative of expression of CDX1 and isevidence that the individual may be suffering from metastasized coloncancer or primary or metastatic stomach and/or esophageal cancer. Inpatients suspected of suffering from colorectal cancer, the presence ofCDX1 protein or of the CDX1 gene transcript in samples outside thecolorectal track is supportive of the conclusion that the individual issuffering from metastatic colorectal cancer. The diagnosis of metastaticcolorectal cancer may be made or confirmed. In patients suspected ofsuffering from stomach or esophageal cancer, the presence of CDX1protein or of the CDX1 gene transcript in samples outside the colorectaltrack is supportive of the conclusion that the individual is sufferingfrom primary and/or metastatic stomach or esophageal cancer. Thediagnosis of primary and/or metastatic stomach or esophageal cancer maybe made or confirmed.

The invention further relates to in vitro methods of determining whetheror not tumor cells are colorectal, stomach or esophageal in origin. Thepresent invention relates to in vitro methods of diagnosing whether ornot an individual suffering from cancer is suffering from colorectal,stomach or esophageal cancer. The present invention relates to in vitromethods of examining samples of tumors from an individual to determinewhether or not CDX1 protein, which is expressed by colorectal, stomachor esophageal tumor cells, is being expressed by the tumor cells. Thepresence of a CDX1 protein or of the CDX1 gene transcript is indicativeof expression of CDX1 and evidence that the individual may be sufferingfrom colorectal, stomach or esophageal cancer. In tumors which aresuspected of being colorectal, stomach or esophageal tumors, thepresence of a CDX1 protein or of the CDX1 gene transcript supports theconclusion that the tumors are of colorectal, stomach or esophagealcancer and the diagnosis of colorectal, stomach or esophageal cancer.

The invention further relates to in vitro kits for practicing themethods of the invention and to reagents and compositions useful ascomponents in such in vitro kits of the invention.

The invention further relates to a method of imaging primary andmetastatic stomach and esophageal tumors and metastatic colorectaltumors and to methods of treating an individual suspected of sufferingfrom primary and metastatic stomach and esophageal tumors and metastaticcolorectal tumors comprising the steps of administering to saidindividual a pharmaceutical compositions according to the invention,wherein the compositions or conjugated compounds are present in anamount effective for therapeutic or diagnostic use in humans sufferingfrom primary and/or metastatic stomach or esophageal tumors andmetastatic colorectal tumors cancer.

The invention further relates to a method of delivering an active agentto primary and metastatic stomach and esophageal tumor cells andmetastatic colorectal tumors cells comprising the steps of administeringto an individual who has primary and/or metastatic stomach or esophagealtumors or metastatic colorectal cancer, a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier or diluent, and anunconjugated compositions that comprises a liposome that includes CDX1ligands on its surface and an active component encapsulated therein.

The invention further relates to killed or inactivated colorectal,stomach or esophageal tumor cells that comprise a protein comprising atleast one epitope of a CDX1 protein; and to vaccines comprising thesame. In some embodiments, the killed or inactivated cells or particlescomprise a CDX1 protein. In some embodiments, the killed or inactivatedcells or particles are haptenized.

The invention further relates to methods of treating individualssuffering from colorectal, stomach or esophageal cancer and to methodsof treating individuals susceptible colorectal, stomach or esophagealcancer. The method of the present invention provides administering tosuch individuals an effective amount of such vaccines. The inventionfurther relates to the use of such vaccines as immunotherapeutics.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Definitions

As used herein, the term “CDX1” is meant to refer to the cellularprotein expressed by normal colorectal cells, as well as primary andmetastasized colorectal, stomach and esophageal cancer cells. In normalindividuals, CDX1 is found exclusively in cells of intestine, inparticular in cells in the duodenum, small intestine (jejunum andileum), the large intestine, colon (cecum, ascending colon, transversecolon, descending colon and sigmoid colon) and rectum.

As used herein, the term “functional fragment” as used in the term“functional fragment of a CDX1 gene transcript” is meant to refer tofragments of CDX1 gene transcript which are functional with respect tonucleic acid molecules with full length sequences. For example, afunctional fragment may be useful as an oligonucleotide or nucleic acidprobe, a primer, an antisense oligonucleotide or nucleic acid moleculeor a coding sequence. The nucleotide sequence encoding human CDX1protein is disclosed in GenBank Accession No. XM 003791, which isincorporated herein by reference.

As used herein, the term “functional fragment” as used in the term“functional fragment of a CDX1 protein” is meant to fragments of CDX1protein which function in the same manner as CDX1 protein with fulllength sequences. For example, an immunogenically functional fragment ofa CDX1 protein comprises an epitope recognized by an anti-CDX1 antibody.A ligand-binding functional fragment of CDX1 comprises a sequence whichforms a structure that can bind to a ligand which recognizes and bindsto CDX1 protein.

As used herein, the term “epitope recognized by an anti-CDX1 proteinantibody” refers to those epitopes specifically recognized by ananti-CDX1 protein antibody.

As used herein, the term “antibody” is meant to refer to complete,intact antibodies, and Fab fragments and F(ab)₂ fragments thereof.Complete, intact antibodies include monoclonal antibodies such as murinemonoclonal antibodies, chimeric antibodies and humanized antibodies.

As used herein, the term “CDX1 ligand” is meant to refer to compoundswhich specifically bind to a CDX1 protein. Antibodies that bind to CDX1are CDX1 ligands. A CDX1 ligand may be a protein, peptide or anon-peptide.

As used herein, the term “active agent” is meant to refer to compoundsthat are therapeutic agents or imaging agents.

As used herein, the term “radiostable” is meant to refer to compoundswhich do not undergo radioactive decay; i.e. compounds which are notradioactive.

As used herein, the term “therapeutic agent” is meant to refer tochemotherapeutics, toxins, radiotherapeutics, targeting agents orradiosensitizing agents.

As used herein, the term “chemotherapeutic” is meant to refer tocompounds that, when contacted with and/or incorporated into a cell,produce an effect on the cell including causing the death of the cell,inhibiting cell division or inducing differentiation.

As used herein, the term “toxin” is meant to refer to compounds that,when contacted with and/or incorporated into a cell, produce the deathof the cell.

As used herein, the term “radiotherapeutic” is meant to refer toradionuclides which when contacted with and/or incorporated into a cell,produce the death of the cell.

As used herein, the term “targeting agent” is meant to refer compoundswhich can be bound by and or react with other compounds. Targetingagents may be used to deliver chemotherapeutics, toxins, enzymes,radiotherapeutics, antibodies or imaging agents to cells that havetargeting agents associated with them and/or to convert or otherwisetransform or enhance co-administered active agents. A targeting agentmay include a moiety that constitutes a first agent that is localized tothe cell which when contacted with a second agent either is converted toa third agent which has a desired activity or causes the conversion ofthe second agent into an agent with a desired activity. The result isthe localized agent facilitates exposure of an agent with a desiredactivity to the cancer cell.

As used herein, the term “radiosensitizing agent” is meant to refer toagents which increase the susceptibility of cells to the damagingeffects of ionizing radiation. A radiosensitizing agent permits lowerdoses of radiation to be administered and still provide atherapeutically effective dose.

As used herein, the term “imaging agent” is meant to refer to compoundswhich can be detected.

As used herein, the term “CDX1 binding moiety” is meant to refer to theportion of a conjugated compound that constitutes an CDX1 ligand. Asused herein, the term “active moiety” is meant to refer to the portionof a conjugated compound that constitutes an active agent.

As used herein, the terms “conjugated compound” and “conjugatedcomposition” are used interchangeably and meant to refer to a compoundwhich comprises a CDX1 binding moiety and an active moiety and which iscapable of binding to CDX1. Conjugated compounds according to thepresent invention comprise a portion which constitutes an CDX1 ligandand a portion which constitutes an active agent. Thus, conjugatedcompounds according to the present invention are capable of specificallybinding to the CDX1 and include a portion which is a therapeutic agentor imaging agent. Conjugated compositions may comprise crosslinkersand/or molecules that serve as spacers between the moieties.

As used herein, the terms “crosslinker”, “crosslinking agent”,“conjugating agent”, “coupling agent”, “condensation reagent” and“bifunctional crosslinker” are used interchangeably and are meant torefer to molecular groups which are used to attach the CDX1 ligand andthe active agent to thus form the conjugated compound.

As used herein, the term “colorectal cancer” is meant to include thewell-accepted medical definition that defines colorectal cancer as amedical condition characterized by cancer of cells of the intestinaltract below the small intestine (i.e. the large intestine (colon),including the cecum, ascending colon, transverse colon, descendingcolon, and sigmoid colon, and rectum). Additionally, as used herein, theterm “colorectal cancer” is meant to further include medical conditionswhich are characterized by cancer of cells of the duodenum and smallintestine (jejunum and ileum). The definition of colorectal cancer usedherein is more expansive than the common medical definition but isprovided as such since the cells of the duodenum and small intestinealso contain CDX1.

As used herein, the term “stomach cancer” is meant to include thewell-accepted medical definition that defines stomach cancer as amedical condition characterized by cancer of cells of the stomach.

As used herein, the term “esophageal cancer” is meant to include thewell-accepted medical definition that defines esophageal cancer as amedical condition characterized by cancer of cells of the esophagus. Asused herein, the term “metastasis” is meant to refer to the process inwhich cancer cells originating in one organ or part of the body relocateto another part of the body and continue to replicate. Metastasizedcells subsequently form tumors which may further metastasize. Metastasisthus refers to the spread of cancer from the part of the body where itoriginally occurs to other parts of the body.

As used herein, the term “metastasized colorectal cancer cells” is meantto refer to colorectal cancer cells which have metastasized.Metastasized colorectal cancer cells localized in a part of the bodyother than the duodenum, small intestine (jejunum and ileum), largeintestine (colon), including the cecum, ascending colon, transversecolon, descending colon, and sigmoid colon, and rectum.

As used herein, the term “metastasized stomach cancer cells” is meant torefer to stomach cancer cells which have metastasized. Metastasizedstomach cancer cells localized in a part of the body other than thestomach.

As used herein, the term “metastasized esophageal cancer cells” is ismeant to refer to colorectal cancer cells which have metastasized.Metastasized esophageal cancer cells localized in a part of the bodyother than the esophagus.

As used herein, the term “non-colorectal sample” and “extra-intestinalsample” are used interchangeably and meant to refer to a sample oftissue or body fluid from a source other than colorectal tissue. In somepreferred embodiments, the non-colorectal sample is a sample of tissuesuch as lymph nodes. In some preferred embodiments, the non-colorectalsample is a sample of extra-intestinal tissue which is an adenocarcinomaof unconfirmed origin. In some preferred embodiments, the non-colorectalsample is a blood sample.

As used herein, “an individual suffering from an adenocarcinoma ofunconfirmed origin” is meant to refer to an individual who has a tumorin which the origin has not been definitively identified.

As used herein, “an individual is suspected of being susceptible tocolorectal, stomach or esophageal cancer” is meant to refer to anindividual who is at a particular risk of developing colorectal, stomachor esophageal cancer. Examples of individuals at a particular risk ofdeveloping colorectal, stomach or esophageal cancer are those whosefamily medical history indicates above average incidence of colorectal,stomach or esophageal cancer among family members and/or those who havealready developed colorectal, stomach or esophageal cancer and have beeneffectively treated who therefore face a risk of relapse and recurrence.

As used herein, the term “antisense composition” and “antisensemolecules” are used interchangeably and are meant to refer to compoundsthat regulate transcription or translation by hybridizing to DNA or RNAand inhibiting and/or preventing transcription or translation fromtaking place. Antisense molecules include nucleic acid molecules andderivatives and analogs thereof. Antisense molecules hybridize to DNA orRNA in the same manner as complementary nucleotide sequences doregardless of whether or not the antisense molecule is a nucleic acidmolecule or a derivative or analog. Antisense molecules may inhibit orprevent transcription or translation of genes whose expression is linkedto cancer.

As used herein, the term “CDX1 immunogen” is meant to refer to CDX1protein or a fragment thereof or a protein that comprises the same or ahaptenized product thereof, cells and particles which display at leastone CDX1 epitope, and haptenized cells and haptenized particles whichdisplay at least one CDX1 epitope.

As used herein, the term “recombinant expression vector” is meant torefer to a plasmid, phage, viral particle or other vector which, whenintroduced into an appropriate host, contains the necessary geneticelements to direct expression of the coding sequence that encodes theprotein. The coding sequence is operably linked to the necessaryregulatory sequences. Expression vectors are well known and readilyavailable. Examples of expression vectors include plasmids, phages,viral vectors and other nucleic acid molecules or nucleic acid moleculecontaining vehicles useful to transform host cells and facilitateexpression of coding sequences.

As used herein, the term “illegitimate transcription” is meant to referto the low level or background expression of tissue-specific genes incells from other tissues. The phenomenon of illegitimate transcriptionthus provides copies of mRNA for a tissue specific transcript in othertissues. If detection techniques used to detect gene expression aresufficiently sensitive to detect illegitimate transcription, theexpression level of the transcript in negative samples due toillegitimate transcription must be discounted using controls and/orquantitative assays and/or other means to eliminate the incidence offalse positive due to illegitimate transcription. Alternatively,detection of evidence of CDX1 gene expression in sample is achievedwithout detecting CDX1 gene transcript present due to illegitimatetranscription. This is accomplished using techniques which are notsufficiently sensitive to detect the CDX1 gene transcript present due toillegitimate transcription which is present as background.

CDX1

Carcinomas derived from the colorectal cells, stomach or esophagusexpress CDX1. The expression of CDX1 by such tumors enables this proteinand its mRNA to be a specific biomarker for the presence of cancer cellsin extra-intestinal tissues and blood. Indeed, this characteristicpermits the detection of CDX1 mRNA by RT-PCR analysis to be a diagnostictest to stage patients with colorectal, stomach or esophageal cancer andfollow patients after surgery for evidence of recurrent disease in theirblood as well as to detect colorectal, stomach and esophageal cancers.Further, the CDX1 may be targeted with a ligand conjugated to an activeagent in order to deliver the active agent to tumor cells in vivo.

U.S. Pat. No. 5,518,888 issued May 21, 1996 to Waldman, PCT applicationPCT/US94/12232 filed Oct. 26, 1994, U.S. application Ser. No. 08/467,920filed Jun. 6, 1995, and U.S. application Ser. No. 08/583,447 filed Jan.5, 1996, which are each incorporated herein by reference, disclose thatmetastasized colorectal tumors can be targeted for delivery of activecompounds by targeting ST receptors (also referred to as guanylincyclase C or GCC). The presence of ST receptors on cells outside of theintestinal tract as a marker for colorectal cancer allows for thescreening, identification and treatment of individuals with metastasizedcolorectal tumors. ST receptors may also be used to target delivery ofgene therapeutics and antisense compounds to colorectal cells.

U.S. Pat. No. 5,601,990 issued Feb. 11, 1997 to Waldman, PCT applicationPCT/US94/12232 filed Oct. 26, 1994, and PCT application PCT/US97/07467filed May 2, 1997, which are each incorporated herein by reference,disclose that detection of evidence of expression of ST receptors insamples of tissue and body fluid from outside the intestinal trackindicate metastasized colorectal cancer.

PCT application PCT/US97/07565 filed May 2, 1997, which is incorporatedherein by reference, disclose that immunogens with epitopes that can betargeted by antibodies that react with ST receptors can be used invaccines compositions useful as prophylactic and therapeuticanti-metastatic colorectal cancer compositions.

It has been discovered that in addition to normal colon cells, primaryand metastasized colon, stomach and esophageal carcinoma cells expressCDX1. Normal stomach and esophageal cells do not express CDX1. Thus, thepresent invention provides the use of CDX1 as a specific moleculardiagnostic marker for the diagnosis, staging, and post-operativesurveillance of patients with metastasized colon cancer and primary andmetastasized stomach and esophageal cancer.

Detection of the expression of CDX1 employing molecular techniques,including, but not limited to, RT-PCR, can be employed to diagnose andstage patients, follow the development of recurrence after surgeryand/or remission, and, potentially, screen normal people for thedevelopment of colorectal, stomach or esophageal cancer.

The amino acid of the CDX1 protein and the nucleotide sequence of theCDX1 gene transcript is set forth in GenBank Accession No. XM 003791,which is incorporated herein by reference.

CDX1 is unique in that it is only expressed in normal intestinal cells.Mucosal cells lining the intestine are joined together by tightjunctions which form a barrier against the passage of intestinalcontents into the blood stream and components of the blood stream intothe intestinal lumen. Therefore, the apical location of cells expressingCDX1 results in the isolation of such cells from the circulatory systemso that they may be considered to exist separate from the rest of thebody; essentially the “outside” of the body. Therefore, the rest of thebody is considered “outside” the intestinal tract. Compositionsadministered “outside” the intestinal tract are maintained apart andsegregated from the only cells which normally express CDX1. Conversely,tissue samples taken from tissue outside of the intestinal tract do notnormally contain cells which express CDX1.

In individuals suffering from colorectal cancer, the cancer cells areoften derived from cells that produce and display the CDX1 and thesecancer cells continue to produce CDX1. It has been observed that CDX1 isexpressed by colorectal cancer cells. Likewise, CDX1 is expressed bystomach and esophageal cancer cells.

The expression of CDX1 by colorectal tumor cells provides a detectabletarget for in vitro screening, monitoring and staging as well as atarget for in vivo delivery of conjugated compositions that compriseactive agents for the imaging and treatment. CDX1 can also serve astargets for vaccines which may be used to protect against metastasizedcolorectal cancer or to treat individiuals with metastasized colorectalcancer.

The expression of CDX1 by stomach and esophageal tumor cells provides adetectable target for in vitro screening, monitoring and staging as wellas a target for in vivo delivery of conjugated compositions thatcomprise active agents for the imaging and treatment. CDX1 can alsoserve as targets for vaccines which may be used to protect againstprimary and metastatic stomach and esophageal cancer or to treatindividiuals with primary and metastatic stomach and esophageal cancer.

In vitro Diagnostics

According to some embodiments of the invention, compositions, kits andin vitro methods are provided for screening, diagnosing and analyzingpatients and patient samples to detect evidence of CDX1 expression bycells outside of the intestinal tract wherein the expression of CDX1 maybe suggestive of metastasized colorectal cancer or primary or metastaticstomach or esophageal cancer. In patients suspected of havingmetastasized colorectal cancer or primary or metastatic stomach oresophageal cancer evidence of CDX1 expression by cells outside of theintestinal tract is indicative of metastasized colorectal cancer orprimary or metastatic stomach or esophageal cancer and can be used inthe diagnosis, monitoring and staging of such patients. Furthermore, thepresent invention relates to methods, compositions and kits useful inthe in vitro screening, and analysis of patient and patient samples todetect evidence of CDX1 expression by tumor cells outside of theintestinal tract wherein the presence of cells that express CDX1-1suggests or confirms that a tumor is of colorectal or stomach oresophageal cancer origin. In an additional aspect of the invention,compositions, kits and methods are provided which are useful tovisualize metastasized colorectal cancer or primary or metastaticstomach or esophageal cancer cells. In vitro screening and diagnosticcompositions, methods and kits can be used in the monitoring ofindividuals who are in high risk groups for colorectal, stomach oresophageal cancer such as those who have been diagnosed with localizeddisease and/or metastasized disease and/or those who are geneticallylinked to the disease. In vitro screening and diagnostic compositions,methods and kits can be used in the monitoring of individuals who areundergoing and/or have been treated for primary colorectal, stomach oresophageal cancer to determine if the cancer has metastasized. In vitroscreening and diagnostic compositions, methods and kits can be used inthe monitoring of individuals who are undergoing and/or have beentreated for colorectal, stomach or esophageal cancer to determine if thecancer has been eliminated. In vitro screening and diagnosticcompositions, methods and kits can be used in the monitoring ofindividuals who are otherwise susceptible, i.e. individuals who havebeen identified as genetically predisposed such as by genetic screeningand/or family histories. Advancements in the understanding of geneticsand developments in technology as well as epidemiology allow for thedetermination of probability and risk assessment an individual has fordeveloping stomach or esophageal cancer. Using family health historiesand/or genetic screening, it is possible to estimate the probabilitythat a particular individual has for developing certain types of cancerincluding colorectal, stomach or esophageal cancer. Those individualsthat have been identified as being predisposed to developing aparticular form of cancer can be monitored or screened to detectevidence of colorectal, stomach or esophageal cancer. Upon discovery ofsuch evidence, early treatment can be undertaken to combat the disease.Accordingly, individuals who are at risk for developing colorectal,stomach or esophageal cancer may be identified and samples may beisolated form such individuals. The invention is particularly useful formonitoring individuals who have been identified as having family medicalhistories which include relatives who have suffered from colorectal,stomach or esophageal cancer. Likewise, the invention is particularlyuseful to monitor individuals who have been diagnosed as havingcolorectal, stomach or esophageal cancer and, particularly those whohave been treated and had tumors removed and/or are otherwiseexperiencing remission including those who have been treated forcolorectal, stomach or esophageal cancer.

In vitro screening and diagnostic compositions, methods and kits can beused in the analysis of tumors. Expression of CDX1 is a marker for celltype and suggests the origin of adenocarcinoma of unconfirmed origin maybe colorectal, stomach or esophageal tumors. Detection of CDX1expression can also be used to assist in an initial diagnosis ofcolorectal, stomach or esophageal cancer or to confirm such diagnosis.Tumors believed to be colorectal, stomach or esophageal in origin can beconfirmed as such using the compositions, methods and kits of theinvention.

In vitro screening and diagnostic compositions, kits and methods of theinvention can be used to analyze tissue samples from the stomach oresophagus to identify primary stomach or esophageal cancer.

In vitro screening and diagnostic compositions, kits and methods of theinvention can be used to analyze tissue samples from the colon to detectthe amount of invasion by primary colorectal cancer into the intestinaltissue.

According to the invention, compounds are provided which bind to CDX1gene transcript or protein. Normal tissue in the body does not have CDX1transcript or protein except cells of the intestinal tract. Theexpression of CDX1 is a marker for cell type and is useful in theidentification of colorectal, stomach or esophageal cancer inextra-intestinal samples.

In some embodiments of the invention, non-colorectal tissue and fluidsamples or tumor samples may be screened to identify the presence orabsence of CDX1 protein. Techniques such as ELISA assays and Westernblots may be performed to determine whether CDX1 is present in a sample.

In some embodiments of the invention, non-colorectal tissue and fluidsamples or tumor samples may be screened to identify whether CDX1 arebeing expressed in cells outside of the colorectal tract by detectingthe presence or absence of CDX1 gene transcript. The presence of CDX1gene transcript or cDNA generated therefrom can be determined usingtechniques such as PCR amplification, branched oligonucleotidetechnology, Northern Blots (mRNA), Southern Blots (cDNA), oroligonucleotide hybridization.

In some embodiments of the invention, cells of non-colorectal tissuesamples or tumor samples may be examined to identify the presence orabsence of CDX1 proteins. Techniques such as immunohistochemistry blotsmay be performed on tissue sections to determine whether CDX1 arepresent in a sample.

In some embodiments of the invention, cells of non-colorectal tissuesamples or tumor samples may be examined to determine whether CDX1 arebeing expressed in cells outside of the colorectal tract by detectingthe presence or absence of the CDX1 gene transcript. The presence of theCDX1 gene transcript or cDNA generated therefrom in cells from tissuesections can be determined using techniques such as in situhybridization.

The presence of CDX1 in non-colorectal tissue and fluid samples or oncells from non-colorectal tissue samples suggests possible stomach oresophageal cancer. The presence of CDX1 in a tumor sample or on tumorcells suggests that the tumor may be colorectal, stomach or esophagealin origin. The presence of the CDX1 gene transcript in non-colorectaltissue and fluid samples or in cells from non-colorectal tissue samplessuggests possible colorectal, stomach or esophageal cancer. The presenceof the CDX1 gene transcript in tumor samples and tumor cells suggeststhat the tumor may be colorectal, stomach or esophageal in origin.

Samples may be obtained from resected tissue or biopsy materialincluding needle biopsy. Tissue section preparation for surgicalpathology may be frozen and prepared using standard techniques.Immunohistochemistry and in situ hybridization binding assays on tissuesections are performed in fixed cells. Extra-intestinal samples may behomogenized by standard techniques such as sonication, mechanicaldisruption or chemical lysis such as detergent lysis. It is alsocontemplated that tumor samples in body fluids such as blood, urine,lymph fluid, cerebral spinal fluid, amniotic fluid, vaginal fluid, semenand stool samples may also be screened to determine if such tumors arecolorectal, stomach or espophageal in origin.

Non-colorectal tissue samples may be obtained from any tissue exceptthose of the colorectal tract, i.e. the intestinal tract below the smallintestine (i.e. the large intestine (colon), including the cecum,ascending colon, transverse colon, descending colon, and sigmoid colon,and rectum) and additionally the duodenum and small intestine (jejunumand ileum). The normal cells of all tissue except those of thecolorectal tract do not express CDX1. Thus if CDX1 protein or the CDX1gene transcript are detected in non-colorectal samples, the possiblepresence of colorectal, stomach or esophageal cancer cells is suggested.In some preferred embodiments, the tissue samples are lymph nodes.

Tissue samples may be obtained by standard surgical techniques includinguse of biopsy needles. One skilled in the art would readily appreciatethe variety of test samples that may be examined for CDX1 and recognizemethods of obtaining tissue samples.

Tissue samples may be homogenized or otherwise prepared for screeningfor the presence of CDX1 by well known techniques such as sonication,mechanical disruption, chemical lysis such as detergent lysis orcombinations thereof.

Examples of body fluid samples include blood, urine, lymph fluid,cerebral spinal fluid, amniotic fluid, vaginal fluid and semen. In somepreferred embodiments, blood is used as a sample of body fluid. Cellsmay be isolated from fluid sample such as centrifugation. One skilled inthe art would readily appreciate the variety of test samples that may beexamined for CDX1. Test samples may be obtained by such methods aswithdrawing fluid with a syringe or by a swab. One skilled in the artwould readily recognize other methods of obtaining test samples.

In an assay using a blood sample, the blood plasma may be separated fromthe blood cells. The blood plasma may be screened for CDX1 includingtruncated proteins which are released into the blood when one or moreCDX1 are cleaved from or sloughed off from tumor cells. In someembodiments, blood cell fractions are screened for the presence ofcolorectal, stomach or esophageal tumor cells. In some embodiments,lymphocytes present in the blood cell fraction are screened by lysingthe cells and detecting the presence of CDX1 protein or the CDX1 genetranscript which may be present as a result of the presence of anystomach or esophageal tumor cells that may have been engulfed by theblood cell. In some preferred embodiments, CD34+ cells are removed priorto isolation of mRNA from samples using commercially availableimmuno-columns.

Aspects of the present invention include various methods of determiningwhether a sample contains cells that express CDX1 by nucleotidesequence-based molecular analysis to detect the CDX1 gene transcript.Several different methods are available for doing so including thoseusing Polymerase Chain Reaction (PCR) technology, branchedoligonucleotide technology, Northern blot technology, oligonucleotidehybridization technology, and in situ hybridization technology.

The invention relates to oligonucleotide probes and primers used in themethods of identifying the CDX1 gene transcript and to diagnostic kitswhich comprise such components.

The mRNA sequence-based methods for detect the CDX1 gene transcriptinclude but are not limited to polymerase chain reaction technology,branched oligonucleotide technology, Northern and Southern blottechnology, in situ hybridization technology and oligonucleotidehybridization technology.

The methods described herein are meant to exemplify how the presentinvention may be practiced and are not meant to limit the scope ofinvention. It is contemplated that other sequence-based methodology fordetecting the presence of the CDX1 gene transcript in non-colorectalsamples may be employed according to the invention.

A preferred method to detecting the CDX1 gene transcript in geneticmaterial derived from non-colorectal samples uses polymerase chainreaction (PCR) technology. PCR technology is practiced routinely bythose having ordinary skill in the art and its uses in diagnostics arewell known and accepted. Methods for practicing PCR technology aredisclosed in “PCR Protocols: A Guide to Methods and Applications”,Innis, M. A., et al. Eds. Academic Press, Inc. San Diego, Calif. (1990)which is incorporated herein by reference. Applications of PCRtechnology are disclosed in “Polymerase Chain Reaction” Erlich, H. A.,et al., Eds. Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)which is incorporated herein by reference. U.S. Pat. No. 4,683,202, U.S.Pat. No. 4,683,195, U.S. Pat. No. 4,965,188 and U.S. Pat. No. 5,075,216,which are each incorporated herein by reference describe methods ofperforming PCR. PCR may be routinely practiced using Perkin Elmer CetusGENE AMP RNA PCR kit, Part No. N808-0017.

PCR technology allows for the rapid generation of multiple copies of DNAsequences by providing 5′ and 3′ primers that hybridize to sequencespresent in an RNA or DNA molecule, and further providing freenucleotides and an enzyme which fills in the complementary bases to thenucleotide sequence between the primers with the free nucleotides toproduce a complementary strand of DNA. The enzyme will fill in thecomplementary sequences adjacent to the primers. If both the 5′ primerand 3′ primer hybridize to nucleotide sequences on the same smallfragment of nucleic acid, exponential amplification of a specificdouble-stranded size product results. If only a single primer hybridizesto the nucleic acid fragment, linear amplification producessingle-stranded products of variable length.

PCR primers can be designed routinely by those having ordinary skill inthe art using sequence information. The nucleotide sequence of the CDX1gene transcript is set forth in SEQ ID NO:1. To perform this method, RNAis extracted from cells in a sample and tested or used to make cDNAusing well known methods and readily available starting materials. Thosehaving ordinary skill in the art can readily prepare PCR primers. A setof primers generally contains two primers. When performing PCR onextracted mRNA or cDNA generated therefrom, if the CDX1 gene transcriptor cDNA generated therefrom is present, multiple copies of the mRNA orcDNA will be made. If it is not present, PCR will not generate adiscrete detectable product. Primers are generally 8-50 nucleotides,preferably about 15-35 nucleotides, more preferably 18-28 nucleotides,which are identical or complementary to and therefor hybridize to theCDX1 gene transcript or cDNA generated therefrom. In preferredembodiments, the primers are each 15-35 nucleotide, more preferably18-28 nucleotide fragments of SEQ ID NO:1. The primer must hybridize tothe sequence to be amplified. Typical primers are 18-28 nucleotides inlength and are generally have 50% to 60% G+C composition. The entireprimer is preferably complementary to the sequence it must hybridize to.Preferably, primers generate PCR products 100 base pairs to 2000 basepairs. However, it is possible to generate products of 50 to up to 10 kband more. If mRNA is used as a template, the primers must hybridize tomRNA sequences. If cDNA is used as a template, the primers musthybridize to cDNA sequences.

The mRNA or cDNA is combined with the primers, free nucleotides andenzyme following standard PCR protocols. The mixture undergoes a seriesof temperature changes. If the CDX1 gene transcript or cDNA generatedtherefrom is present, that is, if both primers hybridize to sequences onthe same molecule, the molecule comprising the primers and theintervening complementary sequences will be exponentially amplified. Theamplified DNA can be easily detected by a variety of well known means.If no CDX1 gene transcript or cDNA generated therefrom is present, noPCR product will be exponentially amplified. The PCR technologytherefore provides an extremely easy, straightforward and reliablemethod of detecting the CDX1 gene transcript in a sample.

PCR product may be detected by several well known means. The preferredmethod for detecting the presence of amplified DNA is to separate thePCR reaction material by gel etectrophoresis and stain the gel withethidium bromide in order to visual the amplified DNA if present. A sizestandard of the expected size of the amplified DNA is preferably run onthe gel as a control.

In some instances, such as when unusually small amounts of RNA arerecovered and only small amounts of cDNA are generated therefrom, it isdesirable or necessary to perform a PCR reaction on the first PCRreaction product. That is, if difficult to detect quantities ofamplified DNA are produced by the first reaction, a second PCR can beperformed to make multiple copies of DNA sequences of the firstamplified DNA. A nested set of primers are used in the second PCRreaction. The nested set of primers hybridize to sequences downstream ofthe 5′ primer and upstream of the 3′ primer used in the first reaction.

The present invention includes oligonucleotide which are useful asprimers for performing PCR methods to amplify the CDX1 gene transcriptor cDNA generated therefrom.

According to the invention, diagnostic kits can be assembled which areuseful to practice methods of detecting the presence of the CDX1 genetranscript or cDNA generated therefrom in non-colorectal samples. Suchdiagnostic kits comprise oligonucleotide which are useful as primers forperforming PCR methods. It is preferred that diagnostic kits accordingto the present invention comprise a container comprising a size markerto be run as a standard on a gel used to detect the presence ofamplified DNA. The size marker is the same size as the DNA generated bythe primers in the presence of the CDX1 gene transcript or cDNAgenerated therefrom. Additional components in some kits includeinstructions for carrying out the assay. Additionally the kit mayoptionally comprise depictions or photographs that represent theappearance of positive and negative results. Positive and negativecontrols may also be provided.

PCR assays are useful for detecting the CDX1 gene transcript inhomogenized tissue samples and cells in body fluid samples. It iscontemplated that PCR on the plasma portion of a fluid sample could beused to detect the CDX1 gene transcript.

Another method of determining whether a sample contains cells expressingCDX1 is by branched chain oligonucleotide hybridization analysis of mRNAextracted from a sample. Branched chain oligonucleotide hybridizationmay be performed as described in U.S. Pat. No. 5,597,909, U.S. Pat. No.5,437,977 and U.S. Pat. No. 5,430,138, which are each incorporatedherein by reference. Reagents may be designed following the teachings ofthose patents and that sequence of the CDX1 gene transcript.

Another method of determining whether a sample contains cells expressingCDX1 is by Northern Blot analysis of mRNA extracted from anon-colorectal sample. The techniques for performing Northern blotanalyses are well known by those having ordinary skill in the art andare described in Sambrook, J. et al., (1989) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. mRNA extraction, electrophoretic separation of the mRNA,blotting, probe preparation and hybridization are all well knowntechniques that can be routinely performed using readily availablestarting material.

The mRNA is extracted using poly dT columns and the material isseparated by electrophoresis and, for example, transferred tonitrocellulose paper. Labeled probes made from an isolated specificfragment or fragments can be used to visualize the presence of acomplementary fragment fixed to the paper. Probes useful to identifymRNA in a Northern Blot have a nucleotide sequence that is complementaryto the CDX1 gene transcript. Those having ordinary skill in the artcould use the sequence information in SEQ ID NO:1 to design such probesor to isolate and clone the CDX1 gene transcript or cDNA generatedtherefrom to be used as a probe. Such probes are at least 15nucleotides, preferably 30-200, more preferably 40-100 nucleotidefragments and may be the entire CDX1 gene transcript.

According to the invention, diagnostic kits can be assembled which areuseful to practice methods of detecting the presence of the CDX1 genetranscript in non-colorectal samples by Northern blot analysis. Suchdiagnostic kits comprise oligonucleotide which are useful as probes forhybridizing to the mRNA. The probes may be radiolabeled. It is preferredthat diagnostic kits according to the present invention comprise acontainer comprising a size marker to be run as a standard on a gel. Itis preferred that diagnostic kits according to the present inventioncomprise a container comprising a positive control which will hybridizeto the probe. Additional components in some kits include instructionsfor carrying out the assay. Additionally the kit may optionally comprisedepictions or photographs that represent the appearance of positive andnegative results.

Northern blot analysis is useful for detecting the CDX1 gene transcriptin homogenized tissue samples and cells in body fluid samples. It iscontemplated that PCR on the plasma portion of a fluid sample could beused to detect the CDX1 gene transcript.

Another method of detecting the presence of the CDX1 gene transcript byoligonucleotide hybridization technology. Oligonucleotide hybridizationtechnology is well known to those having ordinary skill in the art.Briefly, detectable probes which contain a specific nucleotide sequencethat will hybridize to nucleotide sequence of the CDX1 gene transcript.RNA or cDNA made from RNA from a sample is fixed, usually to filterpaper or the like. The probes are added and maintained under conditionsthat permit hybridization only if the probes fully complement the fixedgenetic material. The conditions are sufficiently stringent to wash offprobes in which only a portion of the probe hybridizes to the fixedmaterial. Detection of the probe on the washed filter indicatecomplementary sequences.

Probes useful in oligonucleotide assays at least 18 nucleotides ofcomplementary DNA and may be as large as a complete complementarysequence to the CDX1 gene transcript. In some preferred embodiments theprobes of the invention are 30-200 nucleotides, preferably 40-100nucleotides.

One having ordinary skill in the art, using the sequence informationdisclosed in SEQ ID NO:1 can design probes useful in the invention.Hybridization conditions can be routinely optimized to minimizebackground signal by non-fully complementary hybridization. In somepreferred embodiments, the probes are full length clones. Probes are atleast 15 nucleotides, preferably 30-200, more preferably 40-100nucleotide fragments and may be the entire CDX1 gene transcript.

The present invention includes labeled oligonucleotide which are usefulas probes for performing oligonucleotide hybridization. The labeledprobes of the present invention are labeled with radiolabelednucleotides or are otherwise detectable by readily availablenonradioactive detection systems.

According to the invention, diagnostic kits can be assembled which areuseful to practice oligonucleotide hybridization methods of theinvention. Such diagnostic kits comprise a labeled oligonucleotide whichencodes portions of the CDX1 gene transcript. It is preferred thatlabeled probes of the oligonucleotide diagnostic kits according to thepresent invention are labeled with a radionucleotide. Theoligonucleotide hybridization-based diagnostic kits according to theinvention preferably comprise DNA samples that represent positive andnegative controls. A positive control DNA sample is one that comprises anucleic acid molecule which has a nucleotide sequence that is fullycomplementary to the probes of the kit such that the probes willhybridize to the molecule under assay conditions. A negative control DNAsample is one that comprises at least one nucleic acid molecule, thenucleotide sequence of which is partially complementary to the sequencesof the probe of the kit. Under assay conditions, the probe will nothybridize to the negative control DNA sample. Additional components insome kits include instructions for carrying out the assay. Additionallythe kit may optionally comprise depictions or photographs that representthe appearance of positive and negative results.

Oligonucleotide hybridization techniques are useful for detecting theCDX1 gene transcript in homogenized tissue samples and cells in bodyfluid samples. It is contemplated that PCR on the plasma portion of afluid sample could be used to detect the CDX1 gene transcript.

The present invention relates to in vitro kits for evaluating samples oftumors to determine whether or not they are colorectal, stomach oresophageal in origin and to reagents and compositions useful to practicethe same. In some embodiments of the invention, tumor samples may beisolated from individuals undergoing or recovery from surgery to removetumors in the colorectal, stomach or esophagus, tumors in other organsor biopsy material. The tumor sample is analyzed to identify thepresence or absence of the CDX1 gene transcript. Techniques such asimmunohistochemistry assays may be performed to determine whether CDX1are present in cells in the tumor sample. The presence of mRNA thatencodes the CDX1 protein or cDNA generated therefrom can be determinedusing techniques such as in situ hybridization, immunohistochemistry andin situ ST binding assay.

In situ hybridization technology is well known by those having ordinaryskill in the art. Briefly, cells are fixed and detectable probes whichcontain a specific nucleotide sequence are added to the fixed cells. Ifthe cells contain complementary nucleotide sequences, the probes, whichcan be detected, will hybridize to them.

Probes useful in oligonucleotide assays at least 18 nucleotides ofcomplementary DNA and may be as large as a complete complementarysequence to the CDX1 gene transcript. In some preferred embodiments theprobes of the invention are 30-200 nucleotides, preferably 40-100nucleotides.

One having ordinary skill in the art, using the sequence information setforth in SEQ ID NO:1 can design probes useful in in situ hybridizationtechnology to identify cells that express CDX1. Probes preferablyhybridizes to a nucleotide sequence that corresponds to the CDX1 genetranscript. Hybridization conditions can be routinely optimized tominimize background signal by non-fully complementary hybridization.Probes preferably hybridize to the full length CDX1 gene transcript.Probes are at least 15 nucleotides, preferably 30-200, more preferably40-100 nucleotide fragments and may be the CDX1 gene transcript, morepreferably 18-28 nucleotide fragments of the CDX1 gene transcript.

The probes are fully complementary and do not hybridize well topartially complementary sequences. For in situ hybridization accordingto the invention, it is preferred that the probes are detectable byfluorescence. A common procedure is to label probe with biotin-modifiednucleotide and then detect with fluorescently tagged avidin. Hence,probe does not itself have to be labeled with florescent but can besubsequently detected with florescent marker.

The present invention includes labeled oligonucleotide which are usefulas probes for performing oligonucleotide hybridization. That is, theyare fully complementary with mRNA sequences but not genomic sequences.The labeled probes of the present invention are labeled withradiolabeled nucleotides or are otherwise detectable by readilyavailable nonradioactive detection systems.

The present invention relates to probes useful for in situ hybridizationto identify cells that express CDX1.

Cells are fixed and the probes are added to the genetic material. Probeswill hybridize to the complementary nucleic acid sequences present inthe sample. Using a fluorescent microscope, the probes can be visualizedby their fluorescent markers.

According to the invention, diagnostic kits can be assembled which areuseful to practice in situ hybridization methods of the invention arefully complementary with mRNA sequences but not genomic sequences. Forexample, the mRNA sequence includes different exon sequences. It ispreferred that labeled probes of the in situ diagnostic kits accordingto the present invention are labeled with a fluorescent marker.

Immunohistochemistry techniques may be used to identify and essentiallystain cells with CDX1. Such “staining” allows for analysis of metastaticmigration. Anti-CDX1 antibodies such as those described above ofcontacted with fixed cells and the CDX1 present in the cells reacts withthe antibodies. The antibodies are detectably labeled or detected usinglabeled second antibody or protein A to stain the cells.

The techniques described herein for evaluating tumor sections can alsobe used to analyze tissue sections for samples of lymph nodes as well asother tissues to identify the presence of cells that express CDX1. Thesamples can be prepared and “stained” to detect expression of CDX1.

Immunoassay methods may be used in the diagnosis of individualssuffering from colorectal, stomach or esophageal cancer by detectingpresence of CDX1 in sample of non-colorectal tissue or body fluid froman individuals suspected of having or being susceptible to colorectal,stomach or esophageal cancer using antibodies which were produced inresponse to exposure to such CDX1 protein. Moreover, immunoassay methodsmay be used to identify individuals suffering from colorectal, stomachor esophageal cancer by detecting presence of CDX1 in sample of tumorusing antibodies which were produced in response to exposure to suchCDX1 protein.

The antibodies are preferably monoclonal antibodies. The antibodies arepreferably raised against CDX1 made in human cells. Immunoassays arewell known and there design may be routinely undertaken by those havingordinary skill in the art. Those having ordinary skill in the art canproduce monoclonal antibodies which specifically bind to CDX1 and areuseful in methods and kits of the invention using standard techniquesand readily available starting materials. The techniques for producingmonoclonal antibodies are outlined in Harlow, E. and D. Lane, (1988)ANTIBODIES: A Laboratory Manual, Cold Spring Harbor Laboratory, ColdSpring Harbor N.Y., which is incorporated herein by reference, providedetailed guidance for the production of hybridomas and monoclonalantibodies which specifically bind to target proteins. It is within thescope of the present invention to include Fabs, recombinant Fabs,F(Ab)2s, recombinant F(Ab)2s which specifically bind to CDX1 translationproducts in place of antibodies.

Briefly, CDX1 protein is injected into mice. The spleen of the mouse isremoved, the spleen cells are isolated and fused with immortalized mousecells. The hybrid cells, or hybridomas, are cultured and those cellswhich secrete antibodies are selected. The antibodies are analyzed and,if found to specifically bind to the CDX1, the hybridoma which producesthem is cultured to produce a continuous supply of anti-CDX1 specificantibodies.

The antibodies are preferably monoclonal antibodies. The antibodies arepreferably raised against CDX1 made in human cells.

The means to detect the presence of a protein in a test sample areroutine and one having ordinary skill in the art can detect the presenceor absence of a protein or an antibody using well known methods. Onewell known method of detecting the presence of a protein is animmunoassay. One having ordinary skill in the art can readily appreciatethe multitude of ways to practice an immunoassay to detect the presenceof a CDX1 protein in a sample.

According to some embodiments, immunoassays comprise allowing proteinsin the sample to bind a solid phase support such as a plastic surface.Detectable antibodies are then added which selectively binding to theCDX1. Detection of the detectable antibody indicates the presence ofCDX1. The detectable antibody may be a labeled or an unlabeled antibody.Unlabeled antibody may be detected using a second, labeled antibody thatspecifically binds to the first antibody or a second, unlabeled antibodywhich can be detected using labeled protein A, a protein that complexeswith antibodies. Various immunoassay procedures are described inImmunoassays for the 80's, A. Voller et al., Eds., University Park,1981, which is incorporated herein by reference.

Simple immunoassays may be performed in which a solid phase support iscontacted with the test sample. Any proteins present in the test samplebind the solid phase support and can be detected by a specific,detectable antibody preparation. Such a technique is the essence of thedot blot, Western blot and other such similar assays.

Other immunoassays may be more complicated but actually provideexcellent results. Typical and preferred immunometric assays include“forward” assays for the detection of a protein in which a firstanti-protein antibody bound to a solid phase support is contacted withthe test sample. After a suitable incubation period, the solid phasesupport is washed to remove unbound protein. A second, distinctanti-protein antibody is then added which is specific for a portion ofthe specific protein not recognized by the first antibody. The secondantibody is preferably detectable. After a second incubation period topermit the detectable antibody to complex with the specific proteinbound to the solid phase support through the first antibody, the solidphase support is washed a second time to remove the unbound detectableantibody. Alternatively, the second antibody may not be detectable. Inthis case, a third detectable antibody, which binds the second antibodyis added to the system. This type of “forward sandwich” assay may be asimple yes/no assay to determine whether binding has occurred or may bemade quantitative by comparing the amount of detectable antibody withthat obtained in a control. Such “two-site” or “sandwich” assays aredescribed by Wide, Radioimmune Assay Method, Kirkham, Ed., E. & S.Livingstone, Edinburgh, 1970, pp. 199-206, which is incorporated hereinby reference.

Other types of immunometric assays are the so-called “simultaneous” and“reverse” assays. A simultaneous assay involves a single incubation stepwherein the first antibody bound to the solid phase support, the second,detectable antibody and the test sample are added at the same time.After the incubation is completed, the solid phase support is washed toremove unbound proteins. The presence of detectable antibody associatedwith the solid support is then determined as it would be in aconventional “forward sandwich” assay. The simultaneous assay may alsobe adapted in a similar manner for the detection of antibodies in a testsample.

The “reverse” assay comprises the stepwise addition of a solution ofdetectable antibody to the test sample followed by an incubation periodand the addition of antibody bound to a solid phase support after anadditional incubation period. The solid phase support is washed inconventional fashion to remove unbound protein/antibody complexes andunreacted detectable antibody. The determination of detectable antibodyassociated with the solid phase support is then determined as in the“simultaneous” and “forward” assays. The reverse assay may also beadapted in a similar manner for the detection of antibodies in a testsample.

The first component of the immunometric assay may be added tonitrocellulose or other solid phase support which is capable ofimmobilizing proteins. The first component for determining the presenceof CDX1 in a test sample is an anti-CDX1 antibody. By “solid phasesupport” or “support” is intended any material capable of bindingproteins. Well-known solid phase supports include glass, polystyrene,polypropylene, polyethylene, dextran, nylon, amylases, natural andmodified celluloses, polyacrylamides, agaroses, and magnetite. Thenature of the support can be either soluble to some extent or insolublefor the purposes of the present invention. The support configuration maybe spherical, as in a bead, or cylindrical, as in the inside surface ofa test tube or the external surface of a rod. Alternatively, the surfacemay be flat such as a sheet, test strip, etc. Those skilled in the artwill know many other suitable “solid phase supports” for bindingproteins or will be able to ascertain the same by use of routineexperimentation. A preferred solid phase support is a 96-well microtiterplate.

To detect the presence of CDX1, detectable anti-CDX1 antibodies areused. Several methods are well known for the detection of antibodies.

One method in which the antibodies can be detectably labeled is bylinking the antibodies to an enzyme and subsequently using theantibodies in an enzyme immunoassay (EIA) or enzyme-linked immunosorbentassay (ELISA), such as a capture ELISA. The enzyme, when subsequentlyexposed to its substrate, reacts with the substrate and generates achemical moiety which can be detected, for example, byspectrophotometric, fluorometric or visual means. Enzymes which can beused to detectably label antibodies include, but are not limited tomalate dehydrogenase, staphylococcal nuclease, delta-5-steroidisomerase, yeast alcohol dehydrogenase, alpha-glycerophosphatedehydrogenase, triose phosphate isomerase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,glucoamylase and acetylcholinesterase. One skilled in the art wouldreadily recognize other enzymes which may also be used.

Another method in which antibodies can be detectably labeled is throughradioactive isotopes and subsequent use in a radioimmunoassay (RIA)(see, for example, Work, T. S. et al., Laboratory Techniques andBiochemistry in Molecular Biology, North Holland Publishing Company,N.Y., 1978, which is incorporated herein by reference). The radioactiveisotope can be detected by such means as the use of a gamma counter or ascintillation counter or by autoradiography. Isotopes which areparticularly useful for the purpose of the present invention are 3H,125I, 131I, 35S, and 14C. Preferably 125I is the isotope. One skilled inthe art would readily recognize other radioisotopes which may also beused.

It is also possible to label the antibody with a fluorescent compound.When the fluorescent-labeled antibody is exposed to light of the properwave length, its presence can be detected due to its fluorescence. Amongthe most commonly used fluorescent labeling compounds are fluoresceinisothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin,o-phthaldehyde and fluorescamine. One skilled in the art would readilyrecognize other fluorescent compounds which may also be used.

Antibodies can also be detectably labeled using fluorescence-emittingmetals such as 152Eu, or others of the lanthanide series. These metalscan be attached to the protein-specific antibody using such metalchelating groups as diethylenetriaminepentaacetic acid (DTPA) orethylenediamine-tetraacetic acid (EDTA). One skilled in the art wouldreadily recognize other fluorescence-emitting metals as well as othermetal chelating groups which may also be used.

Antibody can also be detectably labeled by coupling to achemiluminescent compound. The presence of the chemiluminescent-labeledantibody is determined by detecting the presence of luminescence thatarises during the course of a chemical reaction. Examples ofparticularly useful chemoluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester. One skilled in the art would readily recognize otherchemiluminescent compounds which may also be used.

Likewise, a bioluminescent compound may be used to label antibodies.Bioluminescence is a type of chemiluminescence found in biologicalsystems in which a catalytic protein increases the efficiency of thechemiluminescent reaction. The presence of a bioluminescent protein isdetermined by detecting the presence of luminescence. Importantbioluminescent compounds for purposes of labeling are luciferin,luciferase and aequorin. One skilled in the art would readily recognizeother bioluminescent compounds which may also be used.

Detection of the protein-specific antibody, fragment or derivative maybe accomplished by a scintillation counter if, for example, thedetectable label is a radioactive gamma emitter. Alternatively,detection may be accomplished by a fluorometer if, for example, thelabel is a fluorescent material. In the case of an enzyme label, thedetection can be accomplished by colorometric methods which employ asubstrate for the enzyme. Detection may also be accomplished by visualcomparison of the extent of enzymatic reaction of a substrate incomparison with similarly prepared standards. One skilled in the artwould readily recognize other appropriate methods of detection which mayalso be used.

The binding activity of a given lot of antibodies may be determinedaccording to well known methods. Those skilled in the art will be ableto determine operative and optimal assay conditions for eachdetermination by employing routine experimentation.

Positive and negative controls may be performed in which known amountsof CDX1 proteins and no CDX1 protein, respectively, are added to assaysbeing performed in parallel with the test assay. One skilled in the artwould have the necessary knowledge to perform the appropriate controls.In addition, the kit may comprise instructions for performing the assay.Additionally the kit may optionally comprise depictions or photographsthat represent the appearance of positive and negative results.

CDX1 may be produced as a reagent for positive controls routinely. Oneskilled in the art would appreciate the different manners in which theCDX1 protein may be produced and isolated.

Antibody composition refers to the antibody or antibodies required forthe detection of the protein. For example, the antibody composition usedfor the detection of a CDX1 in a test sample comprises a first antibodythat binds to the CDX1 as well as a second or third detectable antibodythat binds the first or second antibody, respectively.

To examine a test sample for the presence of a CDX1, a standardimmunometric assay such as the one described below may be performed. Afirst anti-CDX1 antibody, which recognizes a specific portion of CDX1,is added to a 96-well microtiter plate in a volume of buffer. The plateis incubated for a period of time sufficient for binding to occur andsubsequently washed with PBS to remove unbound antibody. The plate isthen blocked with a PBS/BSA solution to prevent sample proteins fromnon-specifically binding the microtiter plate. Test sample aresubsequently added to the wells and the plate is incubated for a periodof time sufficient for binding to occur. The wells are washed with PBSto remove unbound protein. Labeled anti-CDX1 antibodies, which recognizeportions of CDX1 not recognized by the first antibody, are added to thewells. The plate is incubated for a period of time sufficient forbinding to occur and subsequently washed with PBS to remove unbound,labeled anti-CDX1 antibody. The amount of labeled and bound anti-CDX1antibody is subsequently determined by standard techniques.

Kits which are useful for the detection of CDX1 in a test samplecomprise a container comprising anti-CDX1 antibodies and a container orcontainers comprising controls. Controls include one control samplewhich does not contain CDX1 and/or another control sample whichcontained the CDX1. The anti-CDX1 antibodies used in the kit aredetectable such as being detectably labeled. If the detectable anti-CDX1antibody is not labeled, it may be detected by second antibodies orprotein A for example which may also be provided in some kits inseparate containers. Additional components in some kits include solidsupport, buffer, and instructions for carrying out the assay.Additionally the kit may optionally comprise depictions or photographsthat represent the appearance of positive and negative results.

The immunoassay is useful for detecting CDX1 in homogenized tissuesamples and body fluid samples including the plasma portion or cells inthe fluid sample.

Western Blots may be useful in assisting the diagnosis os individualssuffering from stomach or esophageal cancer by detecting presence ofCDX1 of non-colorectal tissue or body fluid. Western blots may also beused to detect presence of CDX1 in sample of tumor from an individualsuffering from cancer. Western blots use detectable anti-CDX1-antibodiesto bind to any CDX1 present in a sample and thus indicate the presenceof the receptor in the sample.

Western blot techniques, which are described in Sambrook, J. et al.,(1989) Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., which is incorporated hereinby reference, are similar to immunoassays with the essential differencebeing that prior to exposing the sample to the antibodies, the proteinsin the samples are separated by gel electrophoresis and the separatedproteins are then probed with antibodies. In some preferred embodiments,the matrix is an SDS-PAGE gel matrix and the separated proteins in thematrix are transferred to a carrier such as filter paper prior toprobing with antibodies. Anti-CDX1 antibodies described above are usefulin Western blot methods.

Generally, samples are homogenized and cells are lysed using detergentsuch as Triton-X. The material is then separated by the standardtechniques in Sambrook, J. et al., (1989) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.

Kits which are useful for the detection of CDX1 in a test sample byWestern Blot comprise a container comprising anti-CDX1 antibodies and acontainer or containers comprising controls. Controls include onecontrol sample which does not contain CDX1 and/or another control samplewhich contains CDX1. The anti-CDX1 antibodies used in the kit aredetectable such as being detectably labeled. If the detectable anti-CDX1antibody is not labeled, it may be detected by second antibodies orprotein A for example which may also be provided in some kits inseparate containers. Additional components in some kits includeinstructions for carrying out the assay. Additionally the kit mayoptionally comprise depictions or photographs that represent theappearance of positive and negative results.

Western blots are useful for detecting CDX1 in homogenized tissuesamples and body fluid samples including the plasma portion or cells inthe fluid sample.

Therapeutic and Prophylactic Vaccines

The invention relates to prophylactic and therapeutic vaccines forprotecting individuals against metastasized colorectal cancer cells andprimary and/or metastatic stomach or esophageal cancer cells and fortreating individuals who are suffering from metastasized colorectalcancer cells and primary and/or metastatic stomach or esophageal cancercells.

According to the present invention, CDX1 serves as targets against whicha protective and therapeutic immune response can be induced.Specifically, vaccines are provided which induce an immune responseagainst CDX1. The vaccines of the invention include, but are not limitedto, the following vaccine technologies:

-   -   1) DNA vaccines, i.e. vaccines in which DNA that encodes at        least an epitope from an CDX1 is administered to an individual's        cells where the epitope is expressed and serves as a target for        an immune response;    -   2) infectious vector mediated vaccines such as recombinant        adenovirus, vaccinia, Salmonella, and BCG wherein the vector        carries genetic information that encodes at least an epitope        from an CDX1 protein such that when the infectious vector is        administered to an individual, the epitope is expressed and        serves as a target for an immune response;    -   3) killed or inactivated vaccines which a) comprise either        killed cells or inactivated viral particles that display at        least an epitope from an CDX1 protein and b) when administered        to an individual serves as a target for an immune response;    -   4) haptenized killed or inactivated vaccines which a) comprise        either killed cells or inactivated viral particles that display        at least an epitope from an CDX1 protein, b) are haptenized to        be more immunogenic and c) when administered to an individual        serves as a target for an immune response;    -   5) subunit vaccines which are vaccines that include protein        molecules that include at least an epitope from an CDX1 protein;        and    -   6) haptenized subunit vaccines which are vaccines that a)        include protein molecules that include at least an epitope from        an CDX1 protein and b) are haptenized to be more immunogenic.

The present invention relates to administering to an individual aprotein or nucleic acid molecule that comprises or encodes,respectively, an immunogenic epitope against which a therapeutic andprophylactic immune response can be induced. Such epitopes are generallyat least 6-8 amino acids in length. The vaccines of the inventiontherefore comprise proteins which are at least, or nucleic acids whichencode at least, 6-8 amino acids in length from CDX1 protein. Thevaccines of the invention may comprise proteins which are at least, ornucleic acids which encode at least 10 to about 1000 amino acids inlength. The vaccines of the invention may comprise proteins which are atleast, or nucleic acids which encode at least, about 25 to about 500amino acids in length. The vaccines of the invention may compriseproteins which are at least, or nucleic acids which encode at least,about 50 to about 400 amino acids in length. The vaccines of theinvention may comprise proteins which are at least, or nucleic acidswhich encode at least, about 100 to about 300 amino acids in length.

The present invention relates to compositions for and methods oftreating individuals who are known to have metastasized colorectalcancer cells and primary and/or metastatic stomach or esophageal cancercells. Metastasized colorectal cancer and primary and/or metastaticstomach or esophageal cancer may be diagnosed by those having ordinaryskill in the art using the methods described herein or art acceptedclinical and laboratory pathology protocols. The present inventionprovides an immunotherapeutic vaccine useful to treat individuals whohave been diagnosed as suffering from metastatic colorectal cancer andprimary and/or metastatic stomach or esophageal cancer. Theimmunotherapeutic vaccines of the present invention may be administeredin combination with other therapies.

The present invention relates to compositions for and methods ofpreventing metastatic colorectal cancer and primary and/or metastaticstomach or esophageal cancer in individual is suspected of beingsusceptible to colorectal, stomach or esophageal cancer. Suchindividuals include those whose family medical history indicates aboveaverage incidence of colorectal, stomach or esophageal cancer amongfamily members and/or those who have already developed colorectal,stomach or esophageal cancer and have been effectively treated whotherefore face a risk of relapse and recurrence. Such individualsinclude those which have been diagnosed as having colorectal, stomach oresophageal cancer including localized only or localized and metastasizedcolorectal, stomach or esophageal cancer which has been resected orotherwise treated. The vaccines of the present invention may be tosusceptible individuals prophylactically to prevent and combatmetastatic colorectal cancer and primary and metastatic stomach oresophageal cancer.

The invention relates to compositions which are the active components ofsuch vaccines or required to make the active components, to methods ofmaking such compositions including the active components, and to methodsof making and using vaccines.

The amino acid and nucleotide sequences of the CDX1 is set forth as SEQID NO:1.

The present invention relates to recombinant vectors, includingexpression vectors, that comprise the CDX1 gene transcript or a fragmentthereof. The present invention relates to recombinant vectors, includingexpression vectors that comprise nucleotide sequences that encode a CDX1protein or a functional fragment thereof.

The present invention relates to host cells which comprise such vectorsand to methods of making CDX1 protein using such recombinant cells.

The present invention relates to the isolated CDX1 gene transcript andto the isolated CDX1 proteins and to isolated antibodies specific forsuch protein and to hybridomas which produce such antibodies.

The present invention relates to the isolated CDX1 and functionalfragments thereof. Accordingly, some aspects of the invention relate toisolated proteins that comprise at least one epitope of an CDX1.

Some aspects of the invention relate to the above described isolatedproteins which are haptenized to render them more immunogenic. That is,some aspects of the invention relate to haptenized proteins thatcomprise at least one CDX1 epitope.

Accordingly, some aspects of the invention relate to isolated nucleicacid molecules that encode proteins that comprise at least one CDX1epitope.

Naked DNA vaccines are described in PCT/US90/01515, which isincorporated herein by reference. Others teach the use of liposomemediated DNA transfer, DNA delivery using microprojectiles (U.S. Pat.No. 4,945,050 issued Jul. 31, 1990 to Sanford et al., which isincorporated herein by reference), and DNA delivery usingelectroporation. In each case, the DNA may be plasmid DNA that isproduced in bacteria, isolated and administered to the animal to betreated. The plasmid DNA molecules are taken up by the cells of theanimal where the sequences that encode the protein of interest areexpressed. The protein thus produced provides a therapeutic orprophylactic effect on the animal.

The use of vectors including viral vectors and other means of deliveringnucleic acid molecules to cells of an individual in order to produce atherapeutic and/or prophylactic immunological effect on the individualare similarly well known. Recombinant vaccines that employ vacciniavectors are, for example, disclosed in U.S. Pat. No. 5,017,487 issuedMay 21, 1991 to Stunnenberg et al. which is incorporated herein byreference.

In some cases, tumor cells from the patient are killed or inactivatedand administered as a vaccine product. Berd et al. May 1986 CancerResearch 46:2572-2577 and Berd et al. May 1991 Cancer Research51:2731-2734, which are incorporated herein by reference, describes thepreparation and use of tumor cell based vaccine products. According tosome aspects of the present invention, the methods and techniquesdescribed in Berd et al. are adapted by using colorectal, stomach oresophageal cancer cells instead of melanoma cells.

The manufacture and use of isolated translation products and fragmentsthereof useful for example as laboratory reagents or components ofsubunit vaccines are well known. One having ordinary skill in the artcan isolate the CDX1 gene transcript or the specific portion thereofthat encodes CDX1 or a fragment thereof. Once isolated, the nucleic acidmolecule can be inserted it into an expression vector using standardtechniques and readily available starting materials.

The recombinant expression vector that comprises a nucleotide sequencethat encodes the nucleic acid molecule that encodes CDX1 or a fragmentthereof or a protein that comprises the CDX1 or a fragment thereof. Therecombinant expression vectors of the invention are useful fortransforming hosts to prepare recombinant expression systems forpreparing the isolated proteins of the invention.

The present invention relates to a host cell that comprises therecombinant expression vector that includes a nucleotide sequence thatencodes CDX1 protein or a fragment thereof or an CDX1 or a fragmentthereof. Host cells for use in well known recombinant expression systemsfor production of proteins are well known and readily available.Examples of host cells include bacteria cells such as E. coli, yeastcells such as S. cerevisiae, insect cells such as S. frugiperda,non-human mammalian tissue culture cells chinese hamster ovary (CHO)cells and human tissue culture cells such as HeLa cells.

The present invention relates to a transgenic non-human mammal thatcomprises the recombinant expression vector that comprises a nucleicacid sequence that encodes the proteins of the invention. Transgenicnon-human mammals useful to produce recombinant proteins are well knownas are the expression vectors necessary and the techniques forgenerating transgenic animals. Generally, the transgenic animalcomprises a recombinant expression vector in which the nucleotidesequence that encodes CDX1 or a fragment thereof or a protein thatcomprises CDX1 or a fragment thereof operably linked to a mammary cellspecific promoter whereby the coding sequence is only expressed inmammary cells and the recombinant protein so expressed is recovered fromthe animal's milk.

In some embodiments, for example, one having ordinary skill in the artcan, using well known techniques, insert such DNA molecules into acommercially available expression vector for use in well knownexpression systems such as those described herein.

The expression vector including the DNA that encodes a CDX1 or afunctional fragment thereof or a protein that comprises a CDX1 or afunctional fragment thereof is used to transform the compatible hostwhich is then cultured and maintained under conditions whereinexpression of the foreign DNA takes place. The protein of the presentinvention thus produced is recovered from the culture, either by lysingthe cells or from the culture medium as appropriate and known to thosein the art. The methods of purifying the CDX1 or a fragment thereof or aprotein that comprises the same using antibodies which specifically bindto the protein are well known. Antibodies which specifically bind to aparticular protein may be used to purify the protein from naturalsources using well known techniques and readily available startingmaterials. Such antibodies may also be used to purify the protein frommaterial present when producing the protein by recombinant DNAmethodology. The present invention relates to antibodies that bind to anepitope which is present on one or more CDX1-1 translation products or afragment thereof or a protein that comprises the same. Antibodies thatbind to an epitope which is present on the CDX1 are useful to isolateand purify the protein from both natural sources or recombinantexpression systems using well known techniques such as affinitychromatography. Immunoaffinity techniques generally are described inWaldman et al. 1991 Methods of Enzymol. 195:391-396, which isincorporated herein by reference. Antibodies are useful to detect thepresence of such protein in a sample and to determine if cells areexpressing the protein. The production of antibodies and the proteinstructures of complete, intact antibodies, Fab fragments and F(ab)₂fragments and the organization of the genetic sequences that encode suchmolecules are well known and are described, for example, in Harlow, E.and D. Lane (1988) ANTIBODIES: A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. which is incorporated herein byreference.

In some embodiments of the invention, transgenic non-human animals aregenerated. The transgenic animals according to the invention containnucleotides that encode CDX1 or a fragment thereof or a protein thatcomprises the same under the regulatory control of a mammary specificpromoter. One having ordinary skill in the art using standardtechniques, such as those taught in U.S. Pat. No. 4,873,191 issued Oct.10, 1989 to Wagner and U.S. Pat. No. 4,736,866 issued Apr. 12, 1988 toLeder, both of which are incorporated herein by reference, can producetransgenic animals which produce CDX1 or a fragment thereof or a proteinthat comprises the same. Preferred animals are goats and rodents,particularly rats and mice.

In addition to producing these proteins by recombinant techniques,automated peptide synthesizers may also be employed to produce CDX1 or afragment thereof or a fragment thereof or a protein that comprises thesame. Such techniques are well known to those having ordinary skill inthe art and are useful if derivatives which have substitutions notprovided for in DNA-encoded protein production.

In some embodiments, the protein that makes up a subunit vaccine or thecells or particles of a killed or inactivated vaccine may be haptenizedto increase immunogenicity. In some cases, the haptenization is theconjugation of a larger molecular structure to CDX1 or a fragmentthereof or a protein that comprises the same. In some cases, tumor cellsfrom the patient are killed and haptenized as a means to make aneffective vaccine product. In cases in which other cells, such asbacteria or eukaryotic cells which are provided with the geneticinformation to make and display a CDX1 or a fragment thereof or aprotein that comprises the same, are killed and used as the activevaccine component, such cells are haptenized to increase immunogenicity.Haptenization is well known and can be readily performed.

Methods of haptenizing cells generally and tumor cells in particular aredescribed in Berd et al. May 1986 Cancer Research 46:2572-2577 and Berdet al. May 1991 Cancer Research 51:2731-2734, which are incorporatedherein by reference. Additional haptenization protocols are disclosed inMiller et al. 1976 J. Immunol. 117(5:1):1591-1526.

Haptenization compositions and methods which may be adapted to be usedto prepare haptenized CDX1 immunogens according to the present inventioninclude those described in the following U.S. patents which are eachincorporated herein by reference: U.S. Pat. No. 5,037,645 issued Aug. 6,1991 to Strahilevitz; U.S. Pat. No. 5,112,606 issued May 12, 1992 toShiosaka et al.; U.S. Pat. No. 4,526,716 issued Jul. 2, 1985 to Stevens;U.S. Pat. No. 4,329,281 issued May 11, 1982 to Christenson et al.; andU.S. Pat. No. 4,022,878 issued May 10, 1977 to Gross. Peptide vaccinesand methods of enhancing immunogenicity of peptides which may be adaptedto modify CDX1 immunogens of the invention are also described in Franciset al. 1989 Methods of Enzymol. 178:659-676, which is incorporatedherein by reference. Sad et al. 1992 Immunolology 76:599-603, which isincorporated herein by reference, teaches methods of makingimmunotherapeutic vaccines by conjugating gonadotropin releasing hormoneto diphtheria toxoid. CDX1 immunogens may be similarly conjugated toproduce an immunotherapeutic vaccine of the present invention. MacLeanet al. 1993 Cancer Immunol. Immunother. 36:215-222, which isincorporated herein by reference, describes conjugation methodologiesfor producing immunotherapeutic vaccines which may be adaptable toproduce an immunotherapeutic vaccine of the present invention. Thehapten is keyhole limpet hemocyanin which may be conjugated to a CDX1immunogen.

Vaccines according to some aspects of the invention comprise apharmaceutically acceptable carrier in combination with a CDX1immunogen. Pharmaceutical formulations are well known and pharmaceuticalcompositions comprising such proteins may be routinely formulated by onehaving ordinary skill in the art. Suitable pharmaceutical carriers aredescribed in Remington's Pharmaceutical Sciences, A. Osol, a standardreference text in this field, which is incorporated herein by reference.The present invention relates to an injectable pharmaceuticalcomposition that comprises a pharmaceutically acceptable carrier and aCDX1 immunogen. The CDX1 immunogen is preferably sterile and combinedwith a sterile pharmaceutical carrier.

In some embodiments, for example, CDX1 or a fragment thereof or afragment thereof or a protein that comprises the same can be formulatedas a solution, suspension, emulsion or lyophilized powder in associationwith a pharmaceutically acceptable vehicle. Examples of such vehiclesare water, saline, Ringer's solution, dextrose solution, and 5% humanserum albumin. Liposomes and nonaqueous vehicles such as fixed oils mayalso be used. The vehicle or lyophilized powder may contain additivesthat maintain isotonicity (e.g., sodium chloride, mannitol) and chemicalstability (e.g., buffers and preservatives). The formulation issterilized by commonly used techniques.

An injectable composition may comprise the CDX1 immunogen in a dilutingagent such as, for example, sterile water, electrolytes/dextrose, fattyoils of vegetable origin, fatty esters, or polyols, such as propyleneglycol and polyethylene glycol. The injectable must be sterile and freeof pyrogens.

The vaccines of the present invention may be administered by any meansthat enables the immunogenic agent to be presented to the body's immunesystem for recognition and induction of an immunogenic response.Pharmaceutical compositions may be administered parenterally, i.e.,intravenous, subcutaneous, intramuscular.

Dosage varies depending upon known factors such as the pharmacodynamiccharacteristics of the particular agent, and its mode and route ofadministration; age, health, and weight of the recipient; nature andextent of symptoms, kind of concurrent treatment, frequency oftreatment, and the effect desired. An amount of immunogen is deliveredto induce a protective or therapeutically effective immune response.Those having ordinary skill in the art can readily determine the rangeand optimal dosage by routine methods.

1. An in vitro method of screening an individual for metastaticcolorectal cancer cells or primary and/or metastatic stomach oresophageal cancer cells comprising the steps of examining a sample ofextraintestinal tissue and/or body fluids from an individual todetermine whether CDX1 is being expressed by cells in said samplewherein expression of said CDX1 indicates a possibility of metastaticcolorectal cancer cells or primary and/or metastatic stomach oresophageal cancer cells in said sample.
 2. The method of claim 1 whereinexpression of said CDX1 by said cells is determined by detecting thepresence of CDX1 gene transcription product.
 3. The method of claim 1wherein expression of said CDX1 by said cells is determined bypolymerase chain reaction wherein said sample is contacted with primersthat selectively amplify CDX1 gene transcript or cDNA generatedtherefrom.
 4. The method of claim 1 wherein expression of said CDX1 bysaid cells is determined by immunoassay wherein said sample is contactedwith antibodies that specifically bind to CDX1 gene translation product.5. The method of claim 1 wherein said sample is body fluid.
 6. Themethod of claim 1 wherein said sample is blood.
 7. The method of claim 1wherein said sample is lymphatic tissue and/or fluid.
 8. The method ofclaim 1 wherein said sample is a lymph node sample.
 9. The method ofclaim 1 wherein the individual has previously been diagnosed with havingcolorectal, stomach or esophageal cancer.
 10. The method of claim 1wherein the individual has previously been diagnosed with and treatedfor colorectal, stomach or esophageal cancer
 11. An in vitro method ofscreening an individual for metastatic colorectal cancer cells orprimary and/or metastatic stomach or esophageal cancer cells comprisingthe steps of examining a sample of extraintestinal tissue and/or bodyfluids from an individual to determine whether CDX1 gene transcriptionor translation product is present in said sample wherein the presence ofCDX1 gene transcription or translation product in said sample indicatesthat the individual may have metastatic colorectal cancer cells orprimary and/or metastatic stomach or esophageal cancer cells in saidsample.
 12. The method of claim 10 comprising the steps of examining asample of extraintestinal tissue and/or body fluids from an individualto determine whether CDX1 gene transcription product is present in saidsample.
 13. The method of claim 12 wherein the presence of CDX1 genetranscription product is determined by polymerase chain reaction whereinsaid sample is contacted with primers that selectively amplify CDX1 genetranscript or cDNA generated therefrom.
 14. The method of claim 11wherein the presence of CDX1 gene translation product is determined byimmunoassay wherein said sample is contacted with antibodies thatspecifically bind to CDX1 gene translation product.
 15. The method ofclaim 11 wherein said sample is body fluid.
 16. The method of claim 11wherein said sample is blood.
 17. The method of claim 11 wherein saidsample is lymphatic tissue and/or fluid.
 18. The method of claim 11wherein said sample is a lymph node sample.
 19. The method of claim 11wherein the individual has previously been diagnosed with havingcolorectal, stomach or esophageal cancer.
 20. The method of claim 11wherein the individual has previously been diagnosed with and treatedfor colorectal, stomach or esophageal cancer
 21. An in vitro method ofconfirming that a tumor cell removed from a patient suspected of havingcolorectal, stomach or esophageal cancer cells is a colorectal, stomachor esophageal tumor cell comprising the step of determining whether atumor cell expresses CDX1 wherein expression of CDX1 indicates that thetumor cell is a stomach or esophageal tumor cell.
 22. The method ofclaim 21 wherein expression of CDX1 by said tumor cell is determined bydetecting the presence of CDX1 gene transcription product.
 23. Themethod of claim 21 wherein expression of CDX1 by said tumor cell isdetermined by polymerase chain reaction wherein mRNA from said tumorcell or cDNA generated therefrom is contacted with primers thatselectively amplify CDX1 gene transcript or cDNA generated therefrom.24. The method of claim 21 wherein expression of CDX1 by said tumor cellis determined by immunoassay wherein protein from said tumor cell iscontacted with antibodies that specifically bind to CDX1 genetranslation product.
 25. A method of diagnosing an individual who hasstomach cancer comprising the steps of examining a sample of stomachtissue to detect the presence of CDX1 transcript or translation productwherein the presence of CDX1 transcript or translation product in astomach sample indicates stomach cancer.
 26. The method of claim 25comprising the steps of examining said sample of stomach tissue todetermine whether CDX1 gene transcription product is present in saidsample.
 27. The method of claim 26 wherein the presence of CDX1 genetranscription product is determined by polymerase chain reaction whereinsaid sample is contacted with primers that selectively amplify CDX1 genetranscript or cDNA generated therefrom.
 28. The method of claim 26wherein the presence of CDX1 gene translation product is determined byimmunoassay wherein said sample is contacted with antibodies thatspecifically bind to CDX1 gene translation product.
 29. A method ofdiagnosing an individual who has esophageal cancer comprising the stepsof examining a sample of esophagus tissue to detect the presence of CDX1transcript or translation product wherein the presence of CDX1transcript or translation product in an esophageal sample indicatesesophageal cancer.
 30. The method of claim 29 comprising the steps ofexamining said sample of esophageal tissue to determine whether CDX1gene transcription product is present in said sample.
 31. The method ofclaim 30 wherein the presence of CDX1 gene transcription product isdetermined by polymerase chain reaction wherein said sample is contactedwith primers that selectively amplify CDX1 gene transcript or cDNAgenerated therefrom.
 32. The method of claim 29 wherein the presence ofCDX1 gene translation product is determined by immunoassay wherein saidsample is contacted with antibodies that specifically bind to CDX1 genetranslation product.
 33. A kit for diagnosing an individual who hascolorectal, stomach and/or esophageal cancer comprising either: a) acontainer comprising polymerase chain reaction primers that selectivelyamplify CDX1 gene transcript or cDNA generated therefrom; and one ormore of: a container comprising a positive PCR assay control sample, acontainer comprising a negative PCR assay control sample, instructionsfor obtaining and/or processing a sample, instructions for performing aPCR diagnostic assay, and photographs or illustrations depicting apositive result and/or a negative result of a PCR diagnostic assay; orb) a container comprising antibodies that specifcially bind to CDX1 genetranslation product; and one or more of: a container comprising apositive immunoassay control sample, a container comprising a negativeimmunoassay control sample, instructions for obtaining and/or processinga sample, instructions for performing an immuno diagnostic assay, andphotographs or illustrations depicting a positive result and/or anegative result of an immuno diagnostic assay.